Corn (Zea mays L.) has a monoecious flowering habit. The male and female flowers are separate but develop on the same plant. The staminate (male) flowers are borne in the tassel and the pistillate (female) flowers are borne on the ears. Corn is predominantly cross-pollinated; pollen from any tassel randomly pollinates the silks on the ears of adjacent plants or even its own silks. The average corn tassel produces 25 million pollen grains, and most ears have 500-1200 kernels.
The development of corn varieties in the early 1900s was dependent on mass selection. This technique involves no pollen control; each ear is pollinated by a random mixture of pollen from neighboring plants in the field. Therefore, selection progress is slow. However, this technique is effective for simply inherited characteristics, such as, ear and plant height, ear number, adaptation, maturity, and kernel and ear characteristics. Grain yield improvement by mass selection is more difficult because random pollination involves both good and poor yielding plants. Also, the effects of genotype and environment cannot be separated using mass selection techniques.
Alternatively, corn breeders employ controlled pollination, artificial selection and genetic analysis to develop numerous genetic lines or varieties of corn displaying a plethora of desired traits, e.g., yield potential, maturity time, disease resistance, insect resistance, ear size, plant height, drought tolerance. Established inbred lines are used as starting material for further crossing, selection, and analysis in order to develop additional varieties. G. H. Shull (1909), of the Carnegie Institute, has been given credit for suggesting the development of pure inbred lines in corn. The method of inbred line development involves self-fertilization (selfing) of open-pollinated varieties and selection of homozygous biotypes. Inbred lines are generally not vigorous, and yields are low. Over time, breeders improve the elite older inbred lines through recycling. Also, new lines are developed from synthetics and populations improved by some form of recurrent selection. The resulting inbred lines from these sources have more vigor, tolerate greater stress by increased plant densities, and have increased yields. More specifically, plants that have been self-pollinated and selected for type for many generations become homozygous at almost all gene loci and produce a uniform population of true inbred breeding progeny. A cross between two different homozygous inbred lines (single cross hybrid) produces a uniform population of hybrid plants that may be heterozygous for many gene loci.
Hybrid corn seed is typically produced by a male sterility system incorporating manual or mechanical detasseling. Alternate strips of two corn inbreds are planted in a field, and the pollen-bearing tassels are removed from one of the inbreds (female). Providing that there is sufficient isolation from sources of foreign corn pollen, the ears of the detasseled inbred will be fertilized only from the other inbred (male) and the resulting seed is therefore hybrid and will form hybrid plants.
The laborious, and occasionally unreliable, detasseling process can be avoided by using cytoplasmic male-sterile (CMS) inbreds. Plants of a CMS inbred are male sterile as a result of factors resulting from the cytoplasmic, as opposed to the nuclear, genome. Thus, this characteristic is inherited exclusively through the female parent in corn plants, since only the female provides cytoplasm to the fertilized seed. CMS plants are fertilized with pollen from another inbred that is not male-sterile. Pollen from the second inbred may or may not contribute genes that make the hybrid plants male-fertile. Seed from detasseled fertile corn and CMS produced seed of the same hybrid can be blended to insure that adequate pollen loads are available for fertilization when the hybrid plants are grown.
There are several methods of conferring genetic male sterility available, such as multiple mutant genes at separate locations within the genome that confer male sterility, as disclosed in U.S. Pat. Nos. 4,654,465 and 4,727,219 and chromosomal translocations as described in U.S. Pat. Nos. 3,861,709 and 3,710,511, the disclosures of which are specifically incorporated herein by reference. There are many other methods of conferring genetic male sterility in the art, each with its own benefits and drawbacks. These methods use a variety of approaches such as delivering into the plant a gene encoding a cytotoxic substance associated with a male tissue specific promoter or an antisense system in which a gene critical to fertility is identified and an antisense to that gene is inserted in the plant (EPO 89/3010153.8 and WO 90/08828).
Another system useful in controlling male sterility makes use of gametocides. Gametocides are not a genetic system, but rather a topical application of chemicals. These chemicals effect cells that are critical to male fertility. The application of these chemicals effects fertility in the plants only for the growing season in which the gametocide is applied (see, U.S. Pat. No. 4,936,904 to Carlson, specifically incorporated herein by reference). Application of the gametocide, timing of the application and genotype specificity often limit the usefulness of the approach.
The use of male sterile inbreds is but one factor in the production of corn hybrids. The development of corn hybrids requires, in general, the development of homozygous inbred lines, the crossing of these lines, and the evaluation of the crosses. Pedigree breeding and recurrent selection breeding methods are used to develop inbred lines from breeding populations. Breeding programs combine the genetic backgrounds from two or more inbred lines or various other germplasm sources into breeding pools from which new inbred lines are developed by selfing and selection of desired phenotypes. The new inbreds are crossed with other inbred lines and the hybrids from these crosses are evaluated to determine which of those have commercial potential. Plant breeding and hybrid development are expensive and time-consuming processes.
Pedigree breeding starts with the crossing of two genotypes, each of which may have one or more desirable characteristics that is lacking in the other or which complements the other. If the two original parents do not provide all the desired characteristics, other sources can be included in the breeding population. In the pedigree method, superior plants are selfed and selected in successive generations. In the succeeding generations the heterozygous condition gives way to homogeneous lines as a result of self-pollination and selection. Typically, in the pedigree method of breeding five or more generations of selfing and selection is practiced: F1 to F2; F2 to F3; F3 to F4; F4 to F5, etc.
In particular, a single cross hybrid results from the cross of two inbred lines, each of which has a genotype that complements the genotype of the other. The hybrid progeny of the first generation is designated F1 and exhibit hybrid vigor, or heterosis, in relation to their inbred parents. Hybrid vigor may be manifested in polygenic traits, such as, increased vegetative growth and increased yield. It is these hybrids that are generally sought in commercial development. That is to say, an objective for commercial corn hybrid development is to produce new inbred lines that combine to produce superior agronomic performance.
Typically, the development of a hybrid corn variety involves three steps: 1) the selection of plants from various germplasm pools for initial breeding crosses; 2) the selfing of the selected plants from the breeding crosses for several generations to produce a series of inbred lines, which, although different from each other, breed true and are highly uniform; and, 3) crossing the selected inbred lines with unrelated inbred lines to produce the hybrid progeny (F1). Once created, a continual supply of hybrid seed can be produced using the inbred parents and the hybrid corn plants can be generated from the hybrid seed.
A single cross hybrid is produced when two inbred lines are crossed to produce the F1 progeny. A double cross hybrid is produced from four inbred lines crossed in pairs (A×B and C×D) and then the two F1 hybrids are crossed again (A×B)×(C×D). Much of the hybrid vigor exhibited by F1 hybrids is lost in the next generation (F2), and consequently, seed from hybrids is not generally used for planting stock.
Hybrid seed production requires elimination or inactivation of pollen produced by the female parent. Incomplete removal or inactivation of the pollen provides the potential for self-pollination. This inadvertently self-pollinated seed may be unintentionally harvested and packaged with hybrid seed. Once the seed is planted, it is possible to identify and select these self-pollinated plants. These self-pollinated plants will be genetically equivalent to the female inbred line used to produce the hybrid. Typically these self-pollinated plants can be identified and selected due to their decreased vigor. Female selfs are identified by their less vigorous appearance for vegetative and/or reproductive characteristics, including shorter plant height, small ear size, ear and kernel shape, cob color, or other characteristics.
Identification of these self-pollinated lines can also be accomplished through molecular marker analyses. See, “The Identification of Female Selfs in Hybrid Maize: A Comparison Using Electrophoresis and Morphology”, Smith, J. S. C. and Wych, R. D., Seed Science and Technology 14, pp. 1-8 (1995), the disclosure of which is expressly incorporated herein by reference. Through these technologies, the homozygosity of the self-pollinated line can be verified by analyzing allelic composition at various loci along the genome. Those methods allow for rapid identification of the invention disclosed herein. See also, “Identification of Atypical Plants in Hybrid Maize Seed by Postcontrol and Electrophoresis” Sarca, V. et al., Probleme de Genetica Teoritca si Aplicata Vol. 20 (1), p. 29-42.
Corn is an important field corp. Thus, a continuing goal of plant breeders is to develop consistently performing, high-yielding corn hybrids that are agronomically sound and based on stable inbred lines. The reasons for this goal are obvious: to maximize the amount of grain produced with the inputs used and minimize susceptibility of the crop to environmental stresses. While approximately 80% of today's corn crop is used to feed livestock, both in the United States and abroad, food and non-food products containing corn number greater than 3500 and are increasing. Corn is also a major source of product for the milling, both dry and wet, industry. Principal products of dry milling include, for example, grits, meal and flour. The principal products of wet milling include, for example, starch, fiber, corn syrup and dextrose. Corn oil recovered from the corn germ is a by-product of both dry and wet milling. Industrial and food applications of wet milling products of corn are based on the general functional and intrinsic properties of corn, such as viscosity, film formation, adhesive properties, taste, protein levels and starch types.
One of these milling products, starch, is comprised of two polymers (polysaccharides), amylose and amylopectin. In particular, starch derived from dent or flint corn is composed of approximately 73% amylopectin and 27% amylose, each of which does not exist free in nature, but as a component of a discrete, semi-crystalline aggregate, called starch granules. Amylose is an essentially linear polymer composed almost entirely of α-1-4 linked D-glucopyranose. Although typically illustrated as a straight chain structure for the sake of simplicity, amylose is actually often helical. The interior of the helix contains hydrogen atoms and is therefore hydrophobic, allowing amylose to form a type of clathrate complex with free fatty acids, fatty acid components of glycerides, some alcohols and iodine. Amylopectin, the predominant molecule in most starches is a branched polymer that is much larger than amylose. Amylopectin is composed of α-1-4 linked glucose segments connected by α-1-6 linked branch points.
Starch bearing plants, e.g., corn, produce different percentages of amylose and amylopectin, different size starch granules and different polymeric weights for both the amylose and amylopectin. The ratio of amylose to amylopectin within a given type of starch is an important consideration with respect to starch functionality in foods; differences in ratios produce markedly different physical and functional properties in the starch. That is, amylose and amylopectin content and structure affect the architecture of the starch granule, gelatinization and pasting profiles, as well as appearance and textural attributes. Heretofore, starch is typically physically and/or chemically treated to manipulate these and other characteristics. For example, for many industrial purposes, the pearl starch fraction is treated with chemicals to make the starch whiter.
Proteins, lipids, moisture and ash (minerals and salts) are also present in starch granules in minute quantities. Starch granule proteins are divided into two types on the basis of their ability to be extracted from the granules: surface and integral. Surface starch granule proteins are extracted with salt solutions, whereas integral starch granule proteins require more rigorous extraction, for example, with the detergent sodium dodecyl sulfate (SDS) or an alkaline solution. Integral proteins are embedded and may be covalently bound in the amylose-amylopectin structure of the granule, while the surface proteins are more loosely associated with the exterior of the granule. In order to separately recover either or both starch or protein components, the protein/starch matrix must be broken. In known processes for separating protein from starch, either steeping destroys the usefulness of protein and starch or the protein is ineffectively recovered.
Typically, corn used for industrial or food purposes is either dry milled or wet milled. Historically, wet milling has been primarily used to process yellow dent corn and the like. Traditionally, white corn varieties are known for their inefficiency or inability to be wet milled. Thus, the corn processing industry relies upon dry milling techniques to process white corn.
The objective of the dry milling process is to remove the bran coat and germ from the corn kernel while keeping the endosperm portion largely intact, a process which, traditionally, has not or has not efficiently been accomplished using a wet milling process. This separation yields prime products high in starch, low in oil, essentially free of bran and germ, and having excellent shelf life and stability. The dry milling process is comprised of several processing steps. Briefly, the incoming grain is cleaned and then moistened, or tempered, to loosen and toughen the bran coat and soften the germ to facilitate separation in the degerminator. The initial separation into the component parts begins in the degerminator, a specially designed attrition mill containing a truncated cone, surfaced with numerous pearling knobs, rotating inside a perforated housing. As the tempered kernels pass through this device, the abrading action peels the bran coat and germ away from the endosperm. The germ, hull, and small endosperm pieces pass through the perforations in the housing (hereinafter referred to as “through stock”) and the larger endosperm pieces exit the end, or tail, of the degerminator (hereinafter referred to as “tail stock”). The remaining steps include aspiration, milling and sifting; and, drying and cooling. The end result is a spectrum of degermed corn products that includes flaking grits, corn flour, corn bran, corn oil and hominy feed. The degermed products are used in a wide variety of food and beverage applications, some of which include breakfast foods, malt beverages, snack food, prepared mixes, batter and breading mixes and low calorie/high fiber foods.
The wet milling process is used primarily to extract starch and gluten from corn. Other fractions that are a part of the by-products include the germ, fiber, and steep water. Generally, wet milling involves an initial water soak under controlled conditions to soften the corn kernels. The corn is then milled and its components separated by screening, centrifuging and washing. The first step in the actual processing is called steeping and includes controlled processing conditions such as temperature, time, and sulfur dioxide concentration. Steeping softens the kernels, facilitating separation of the corn components. The corn kernels are placed in a steeping tank with a countercurrent flow of water at about 120-125° F. The water is treated with sulfur dioxide (SO2) to a concentration of 0.12-0.20% by weight. The sulfur dioxide increases the rate of water diffusion into the kernel and assists in breaking down the protein-starch matrix necessary for high starch yield and quality. The kernels remain in the steep tank for 24-50 hours. In general, 8-9 gallons of water/bushel of corn is required, with about 3.5 gallons/bushel being absorbed by the corn to increase its moisture from approximately 16% to 45% during steeping. The remainder, approximately 4.5-5.5 gallons/bushel, is removed from the system and must be dealt with in an environmentally acceptable manner. The kernels are then dewatered, and subjected to sets of attrition type mills to release the germ.
After the germ is recovered, the remaining kernel components including the starch, hull, fiber, and gluten are subjected to another set of attrition mills and passed through a set of wash screens to separate the fiber components from the starch and gluten. The starch and gluten pass through the screens while the fiber does not. Centrifugation or a third grind followed by centrifugation is used to separate the starch from the protein. Centrifugation produces a slurry which contains the starch granules which is dewatered, washed with fresh water, and dried to about 12% moisture. The amount of extractable starch has been one of the prime concerns of wet millers. It is dependent upon the ease of separation of the components, uniformity of the product, and the hardness of the endosperm. Yellow corn generally has a relatively hard endosperm, requiring a higher level of sulfur dioxide in the steeping process to separate the components. More sulfur dioxide results in more waste by-products requiring disposal.
Traditionally, the dry and wet milling industries have not selected corn hybrids for milling based on particular component profile of the hybrid kernels, for example, protein characteristic of the endosperm, an amino acid characteristic, a β-carotene characteristic, a xanthophyll characteristic, a starch characteristic, and combinations thereof. In fact, it is a common practice in the milling industry to mix together different corn varieties prior to milling in order to obtain more uniform end-products.
There is a need, therefore, to select white corn hybrids for wet milling based on particular grain components, for example, protein characteristic of the endosperm, an amino acid characteristic, a β-carotene characteristic, a xanthophyll characteristic, a starch characteristic, and combinations thereof. There is also a need to select particular white corn hybrids adapted for wet milling and potentially requiring the use of less steeping water and sulfur dioxide during the wet milling process. A further need is for an improved wet milling process for efficiently processing white corn hybrids.
Both environment and genetics affect the properties of corn for alkaline cooking. See, Bedolla, S. 1980, “Effect of genotype on cooking and texture of corn for tortilla production”, M.S. thesis, Texas A&M University, College Station. See also, Goldstein, T. M. 1983, “Effect of environment and genotype on hardness and alkaline cooking properties of maize”, M.S. thesis, Texas A&M University, College Station. In general, the properties desired in corn for alkaline cooking are: uniformly sized kernels of high density and high test weight. A high proportion of hard or flinty endosperm, intact kernels free of fissures or stress cracks, kernels without prominent dents in the crown, easily removed pericarp, clean yellow or white color, and white cobs instead of red.
Rate of cooking is affected by the relative rate of water and alkali uptake by the corn kernels. Improper drying and handling of corn causes fissuring and breakage, which causes overcooking. Soft kernels, broken kernels, or kernels with fissures take up water and alkali more quickly and cook faster. Thus, some kernels are overcooked and may dissolve during handling, which increases dry matter losses, and produces masa with poor properties.
A properly cooked corn kernel consists of enough gelatinized, swollen starch granules and hydrated protein matrix to produce a dough when it is stone-ground. The attrition of the stone disrupts the swollen starch granules and hydrated protein and causes dough formation. The amylose, amylopectin, and protein form a continuous system, i.e., “glue” that holds the ungelatinized starch and intact endosperm cells together in a cohesive dough. Overcooked corn often forms masa with a sticky consistency because too much glue is formed. The complex interaction between amylose, amylopectin, proteins, ungelatinized starch granules, and endosperm particles is not understood. More information on this complex could lead to many practical applications.